ABSTRACT

The
Mycobacterium tuberculosis alternate sigma factor, SigF, is
expressed during stationary growth phase and under stress conditions in
vitro. To better understand the function of SigF we studied the
phenotype of the M. tuberculosis ΔsigF mutant
in vivo during mouse infection, tested the mutant as a vaccine in
rabbits, and evaluated the mutant's microarray expression profile
in comparison with the wild type. In mice the growth rates of theΔ
sigF mutant and wild-type strains were nearly
identical during the first 8 weeks after infection. At 8 weeks, theΔ
sigF mutant persisted in the lung, while the wild
type continued growing through 20 weeks. Histopathological analysis
showed that both wild-type and mutant strains had similar degrees of
interstitial and granulomatous inflammation during the first 12 weeks
of infection. However, from 12 to 20 weeks the mutant strain showed
smaller and fewer lesions and less inflammation in the lungs and
spleen. Intradermal vaccination of rabbits with the M.
tuberculosis ΔsigF strain, followed by aerosol
challenge, resulted in fewer tubercles than did intradermal M.
bovis BCG vaccination. Complete genomic microarray analysis
revealed that 187 genes were relatively underexpressed in the absence
of SigF in early stationary phase, 277 in late stationary phase, and
only 38 genes in exponential growth phase. Numerous regulatory genes
and those involved in cell envelope synthesis were down-regulated in
the absence of SigF; moreover, the ΔsigF mutant strain
lacked neutral red staining, suggesting a reduction in the expression
of envelope-associated sulfolipids. Examination of
5′-untranslated sequences among the downregulated genes
revealed multiple instances of a putative SigF consensus recognition
sequence: GGTTTCX18GGGTAT.
These results indicate that in the mouse the M.
tuberculosis ΔsigF mutant strain persists in the
lung but at lower bacterial burdens than wild type and is attenuated by
histopathologic assessment. Microarray analysis has identified
SigF-dependent genes and a putative SigF consensus recognition
site.

Control of Mycobacterium tuberculosis infection is difficult
due to the complex and long-term nature of the host-pathogen
interactions in this disease. Initial infection is followed
by bacterial multiplication within mononuclear phagocytes, release of
intracellular organisms, and dissemination
(15). The subsequent
development of specific immunity often results in containment of the
infection but not eradication of the organism. Therefore, reactivation
of tuberculosis may occur years after initial exposure
(56). The mechanisms
enabling M. tuberculosis to survive during the late stages of
active infection in the mouse tuberculosis model may have a role in the
development of latent tuberculosis
(54). Disease
pathogenesis in these different stages is likely to involve various
virulence factors which are differentially deployed as well as a system
of genetic adaptation by the pathogen
(32,
47).

In the mouse
tuberculosis model, one key transition point during pathogenesis occurs
at 4 to 8 weeks after infection, as host acquired cell-mediated immune
responses mount (42). At
this time, tubercle bacilli may employ specific adaptive mechanisms
that lead to a state of contained, multibacillary pulmonary infection,
which shares some features of the infection that occur in humans. While
no widely accepted animal model of human latent M.
tuberculosis infection is currently available
(19), the late-stage
multibacillary plateau of lung bacterial counts seen in the mouse has
been proposed to correlate in some aspects with the arrested
paucibacillary state in human latent tuberculosis infection
(32). Consequently, the
identification of bacterial genes required for survival in chronic
murine infection may be valuable for understanding the pathogenesis of
human latent M. tuberculosis infection.

Many studies
have implicated sigma factors in the regulation of virulence gene
expression by M. tuberculosis
(11,
27,
36,
45). The M.
tuberculosis sigF gene was discovered by degenerate PCR
(17) and is a close
homologue of sporulation sigma factors in Streptomyces
coelicolor and Bacillus subtilis as well as stress
response sigma factors in B. subtilis, Staphylococcus
aureus, and Listeria monocytogenes
(16). It is strongly
induced during the stationary phase of growth and under certain stress
conditions, such as nitrogen depletion, cold shock
(17), and exposure to
certain antibiotics (38).
There was no marked change of sigF mRNA expression in M.
tuberculosis H37Rv after a short 2-h exposure to a variety of
stresses in culture (34),
but expression was upregulated during growth within macrophages
(21). Recently, M.
tuberculosis sigF has been shown to be expressed during
nutrient starvation, which may be a model of the nongrowing
drug-resistant state that mimics the persistence of M.
tuberculosis in vivo
(5). The M.
tuberculosis ΔsigF mutant strain, in which the
sigF gene is deleted and replaced by a hygromycin resistance
gene, has been shown to be less virulent in mice by time-to-death
analysis (8).

In
this study, we evaluated the phenotype of the M. tuberculosisΔ
sigF mutant during mouse infection by organ CFU
counts and histopathologic analysis. Finding an attenuated phenotype in
this model, we also tested the efficacy of the ΔsigF
mutant strain as a possible candidate for vaccination against
M. tuberculosis using the rabbit aerosol challenge
model (6,
12). Through microarray
analysis, we studied the global expression of genes under the influence
of SigF during the different stages of in vitro growth. Evaluation of
genes underexpressed in the absence of SigF has permitted the
identification of a putative consensus binding site for
SigF.

MATERIALS AND
METHODS

Bacterial cultivation.The virulent CDC1551 (also known as
CSU93 or Oshkosh) strain of M. tuberculosis
(18,
60) and the Erdman strain
were grown at 37°C on Löwenstein-Jensen medium or in
roller bottles in 7H9-albumin-dextrose complex (7H9-ADC) broth
(Difco Laboratories, Detroit, Mich.) supplemented with 0.2%
glycerol and 0.05% Tween 80. The ΔsigF mutant
strain and the complemented ΔsigF mutant strain were
generated from CDC1551 as described previously
(8). M. bovis BCG
(Pasteur) was maintained under similar conditions. For animal
inoculation, liquid cultures were declumped by brief bath sonication
and settling and were diluted in complete 7H9 medium. Colony counts
from mouse organs were performed by using Middlebrook 7H10-ADC agar
plates, made selective by adding carbenicillin, polymyxin B,
trimethoprim, and amphotericin B to final concentrations of 100μ
g/ml, 200 U/ml, 20 μg/ml, and 10 μg/ml,
respectively.

Mouse virulence
assays.For mouse organ CFU
assays, BALB/c mice (Harlan Sprague Dawley), 6 to 8 weeks old, were
inoculated intravenously with 0.1 ml of dispersed preparations of
mycobacteria. Three inocula were prepared using dispersal techniques as
described previously (40)
and counted on the day of infection. Plate counts showed the actual
inocula to be 6.1 × 105 CFU for wild-type M.
tuberculosis bacteria and 3.6 × 105 CFU for theΔ
sigF mutant strain of M. tuberculosis.
Groups of six mice were sacrificed at weeks 1, 2, 4, 8, 12, 16, and 20.
The lungs and spleen were removed, and the tissues were homogenized in
phosphate-buffered saline-Tween. The homogenates were transferred to
plates with complete 7H10-ADC agar, and the colonies were enumerated to
determine infectious burden in these organs. A similar experiment was
conducted with outbred Swiss-Webster mice, in which the inoculum of theΔ
sigF mutant strain was 3.8 × 105
CFU and that of the wild type was 3.4 × 105
CFU.

Vaccination of rabbits.Strains of ΔsigF
mutant M. tuberculosis CDC1551, M. bovis BCG, and
wild-type M. tuberculosis Erdman were grown to log phase, bead
vortexed, and then allowed to settle. Supernatants were pooled, mixed
with glycerol to a final concentration of 10%, and then frozen
in aliquots at −70°C. The bacterial titers of the
aliquots were determined by plating serial dilutions on Middlebrook
7H10 agar. Vaccine aliquots were thawed and diluted in 10% oleic
acid albumin at the time of vaccination. Pathogen-free New Zealand
White rabbits (2.5 kg each, female) were purchased from Covance
Research Products, Inc. (Denver, Pa.). Animals were vaccinated with 5×
106 organisms intradermally on each flank at time
zero. Nine weeks later, animals were aerosol challenged with M.
tuberculosis Erdman strain (a kind gift of Frank Collins) by a
nose-only manifold system at the U.S. Army Medical Research Institute
of Infectious Diseases, Ft. Detrick, Frederick, Md. Each animal was
exposed for 10 min to an aerosolized 10-ml inoculum of aerosol
containing 106 organisms/ml diluted in 10% oleic acid
albumin. Whole-body plethysmographs and impinger samples of the
aerosols were obtained for each rabbit. The oleic acid-albumin
solutions containing the aerosolized bacteria were cultured at various
dilutions on both Löwenstein-Jensen slants and 7H10 Middlebrook
agar (Fisher). For each rabbit, the number of viable bacilli inhaled
(104 to 105 organisms) was calculated based on
the volume of inhaled air during exposure and the number of CFU per
milliliter cultured from the impinger samples
(6). The animals were
housed in biosafety-level-3 facilities at the George Washington
University Medical Center immediately following infection. After 5
weeks, the rabbits were euthanized with intravenous pentobarbital. The
lungs were removed, and the number and volume of grossly visible
primary tubercles in the lungs were assessed by Lurie's tubercle
count method (14). Lung
specimens for histological examination were fixed in 10%
formalin and paraffin embedded. All animals were maintained in
accordance with protocols approved by the institutional Animal Care and
Use Committee of the three institutions where the work was
performed.

Neutral red
staining.Following growth
on Löwenstein-Jensen medium for 6 weeks, wild-type, knockout, and
complemented strains were washed with 50% methanol at
37°C for 60 min. After decanting and draining the solvent, 5 ml
of 5% NaCl in 0.5% Trizma base, brought to pH 9.5 with 2
N HCl, was added to the cells. For color development, 50 μl of
0.05% neutral red was added, and the washed cells were incubated
for 60 min at 37°C. Neutral red binding was determined by the
color of the cell pellet
(39).

RNA
isolation.Cultures of the
M. tuberculosis ΔsigF mutant strain,
wild-type M. tuberculosis, and the complementedΔ
sigF mutant strain were grown to
A600 values of 0.6, 2.2, and also for 3 days after
an A600 of 2.2 was reached. Total RNA
was extracted from the cultures using Trizol. Briefly, the bacterial
cultures were centrifuged and the pellet washed in phosphate-buffered
saline. The pellet was resuspended in 1 to 3 ml of Trizol along with
0.1-mm-diameter Zirconia/silica microbeads (BioSpec Products,
Bartlesville, Okla.) and agitated on a bead beater three times at 30-s
intervals. Cells were chilled on ice for 1 min following each
disruption. The sample was centrifuged at 14,000 rpm (Eppendorf model
5417R centrifuge), and 200 μl of chloroform/ml of
sample was added to the supernatant, followed by vortexing and
centrifuging for 3 min at 4°C. Isopropanol was added to the
aqueous phase, the RNA precipitated at room temperature, and the pellet
washed with 80% ethanol. After air drying, the pellet was
resuspended in diethyl pyrocarbonate-treated water and stored at−
70°C.

Microarray probe
labeling, hybridization, and analysis.Gene-specific PCR primers were
designed to amplify internal fragments from a total of 4,016 open
reading frames from the annotated sequences of M. tuberculosis
CDC1551 and H37Rv (10,
18,
27). Individual purified
PCR products were spotted in duplicate on High Contact Angle slides
(Corning, Ithaca, N.Y.) using a 96-well format IAS arrayer (Intelligent
Automation Systems, Cambridge, Mass.). Bacterial RNA prepared by the
Trizol method was reverse transcribed and labeled with Cy3 or Cy5
(Amersham Pharmacia) using the aminoallyl labeling method
(27). The slides were
scanned with a Genepix (Axon Instruments, Union City, Calif.) scanner
using Genepix Pro 3.0 software. Spot intensities were defined and
quantified using the TIGR Spotfinder and Array Viewer software systems.
For each bacterial growth point, two independent RNA preparations from
wild type and mutant were prepared. For each RNA sample pair, reverse
labeling was performed by switching the Cy3 and Cy5 dyes (two
hybridizations for each growth point). Each amplicon was spotted in
duplicate, yielding four relative hybridization values for each gene at
each growth point. The ratio of wild type to mutant was determined. Any
ratio greater than 1.7-fold was operationally considered a significant
down-regulation in the ΔsigF mutant strain. In the
same respect, any ratio less than 0.5-fold was considered to be
up-regulated in the ΔsigF mutant strain. The median
value was used for comparison of wild type and
mutant.

RESULTS

Proliferation
and survival of the M. tuberculosis ΔsigF
mutant strain in mouse tissues.We investigated the in vivo growth
phenotype of the M. tuberculosis ΔsigF mutant
strain in the mouse tuberculosis model, which earlier time-to-death
analysis had shown to be less virulent than wild-type
(8). Dispersed
preparations of the ΔsigF mutant strain and wild-type
M. tuberculosis were administered by injection of 3.6×
105 and 6.1 × 105 CFU,
respectively, into the tail veins of groups of BALB/c mice. At the
indicated times, mice were euthanized and the CFU counts were
determined by plating homogenates of the spleen and lung tissues.
Although the initial levels of ΔsigF mutant were lower
at week 1 than those of the wild type, in keeping with the smaller
inoculum given, the in vivo growth rates of the mutant and wild-type
strains were essentially identical from 1 to 8 weeks in lungs (Fig.
1). However, at 8 weeks the M. tuberculosis ΔsigF
mutant strain persisted in the lung while the wild-type strain
continued to proliferate slowly from 8 to 20 weeks (Fig.
1A). At 20 weeks, the
difference in CFU between mutant and wild type was 40-fold in the lung
and 43-fold in the spleen, compared to only 3- and 4-fold differences
at week 1 in the lung and spleen, respectively. This level of
attenuation seen in organ CFU counts is notably greater than that found
with mutant tubercle bacilli lacking either sigH
(27) or sigC
(58) or sigE
(1), which maintained CFU
counts comparable to those of the wild type with reduced pathology. On
the other hand, the ΔsigF mutant strain persists at
considerably higher levels and for a longer period in immunocompetent
mice than do auxotrophic mutants such as the pantothenate auxotroph
(48). A similar
experiment conducted with outbred Swiss-Webster mice, in which the
inoculum of the ΔsigF mutant strain (3.8 ×
105) exceeded that of the wild type (3.4 ×
105) by a factor of 1.1, revealed a similar pattern of
stable lung persistence by the mutant during late-stage infection (data
not shown).

Comparison
of the survival of the ΔsigF mutant strain (○)
and wild-type M. tuberculosis strains (CDC1551) (□) in
the mouse tuberculosis model. Following intravenous
inoculation of mice with 3.6 ×105 (5.56 log) mutant
or 6.1 × 105 (5.79 log) wild-type bacteria, groups
of six mice were sacrificed at the indicated times. A virulence
analysis by whole organ CFU counts was done in the lung
(A) and spleen (B) at each indicated time. Error
bars, standard
deviations.

Histopathological analysis of mouse tissues at the
4th week after infection showed that both wild-type and mutant strains
produced comparable degrees of interstitial inflammation with small,
scattered foci of organizing lesions (Fig.
2A and
D, respectively). Granulomatous inflammation began to form in both strains
at the 12th week. However, wild-type M. tuberculosis displayed
increasing granuloma size, coalescence of the lesions, and progressive
loss of lung parenchyma, while the disease in lungs of mice infected
with the ΔsigF mutant strain showed retarded disease
progression (Fig. 2B and
E, respectively). Indeed, at 20 weeks, lungs of mice
infected with the mutant strain (not shown) showed degrees of
pneumonitis similar to those of wild-type-infected animals at 12 weeks
(Fig. 2B), suggesting a
delay in the development of classic tuberculosis pathology with the
mutant. Acid-fast stains revealed a paucity of bacilli in lung tissues
infected with the ΔsigF mutant strain at 20 weeks
compared with the wild type (Fig. 2C
and F, spleen not shown), which is in agreement with the
organ CFU assay results shown in Fig.
1. At the level of gross
pathology, there were readily apparent differences between tissues
infected by the mutant versus wild type. The organs of animals that
were infected by the mutant strain had smaller and fewer granulomas and
less inflammation than those infected with wild type (e.g., spleen,
Fig. 2G). Hence, theΔ
sigF mutant strain showed a mouse phenotype of
persistent, high-level organ survival but reduced histopathologic
evidence of disease. In contrast, reduced pathology was observed in the
face of equivalent bacterial replication in the M.
tuberculosis ΔsigH mutant
(27).

Microscopic
histopathology of mouse lung tissues during the course of infection by
wild-type M. tuberculosis (A to C) and theΔ
sigF mutant strain (D to F). Low-power views are
representative of lung specimens after formalin fixation, paraffin
embedding, and azure eosin staining, corresponding to an infection
duration of 4 weeks (A and D) and 12 weeks (B and E). Representative
high-power views of lung sections are shown after acid-fast staining at
20 weeks of infection (C and F). Also shown are the unfixed
representative murine spleens (G) at necropsy after infection
for 20 weeks: no infection (left), infection with wild type (middle),
or infection with the ΔsigF mutant strain (right). See
text for
details.

Testing
the efficacy of the ΔsigF mutant strain as a vaccine
in the rabbit model.Since it
has been shown by these and previous data
(8) that the M.
tuberculosis ΔsigF mutant strain is attenuated in
the mouse infection model, we tested this mutant for its efficacy as a
vaccine in the rabbit model. Pathogen-free New Zealand White rabbits,
six per group, were inoculated intradermally with 5 ×
106 CFU of preparations of the ΔsigF mutant
strain and BCG. Five unvaccinated rabbits, matched for age and weight,
were used as controls. No skin lesions formed at the sites of
intradermal vaccination with either the M. tuberculosisΔ
sigF mutant strain or BCG. Nine weeks after
inoculation, the animals were aerosol challenged with the M.
tuberculosis Erdman strain, which is a relatively virulent strain
for rabbits (33). At 5
weeks the rabbits were euthanized and the lungs were removed. The
number and size of the visible primary tubercles were assessed by
Lurie's tubercle count method
(6). Although fewer
tubercles formed in rabbits vaccinated with the ΔsigF
mutant strain compared to those vaccinated with BCG (Table
1), the difference was not statistically significant. The tubercle numbers
in the rabbits vaccinated with the ΔsigF mutant strain
were 56% of the control numbers (P = 0.06 by
one-tailed t test analysis for vaccine-mediated protection,
justified by the expectation, based on the mouse data, and the
observation, in the rabbit data, of no exacerbation of disease caused
by the vaccine strain
[41]), while
BCG-vaccinated rabbits formed 68% of the control numbers
(P = 0.11). The diameters of the tubercles in the
rabbits vaccinated with the ΔsigF mutant strain and
BCG were 76 and 70% of the unvaccinated control animals,
respectively. These results suggest that intradermal vaccination of
rabbits with the M. tuberculosis ΔsigF mutant
strain may offer some degree of protection from tubercle formation in
the rabbit aerosol challenge
model.

Efficacies
of M. tuberculosis ΔsigF mutant strain and
BCG as vaccines in the rabbit aerosol-challenge model and tubercle
count methoda

Microarray identification of
SigF-regulated genes.In
order to identify genes under SigF control or influence, the global
expression patterns of the M. tuberculosis wild-type andΔ
sigF mutant strains were explored at different growth
stages by complete genomic microarrays
(3,
27,
34,
53,
61). Total RNA was
isolated from cultures of the M. tuberculosisΔ
sigF mutant strain and wild-type CDC1551 grown to
A600 values of 0.6, 2.2, and for 3 days after an
A600 of 2.2 was reached (i.e., exponential phase,
early stationary [S] phase, and late S-phase, respectively).
The cDNA from bacterial transcripts was labeled and allowed to
hybridize to a microarray containing 4,016 PCR products, each specific
for a unique M. tuberculosis CDC1551 gene. By examining the
relative intensities (i.e., wild type over mutant) and using an
operational cutoff of ≥1.7-fold, underexpression was found for
38 genes in exponential phase, 187 genes in early S-phase, and 277
genes in late S-phase (Table
2). Of those genes underexpressed in the mutant strain during the three
stages of growth, nearly 50% encode hypothetical proteins or
proteins of unknown function. In addition, there were 68, 22, and 58
relatively overexpressed genes at exponential, early S-, and late
S-phases, respectively, of which ∼40% encode
hypothetical proteins (Table
2). Table
3 lists the most highly down-regulated genes in the
different growth phases, and a complete list of
downregulated genes can be found at the Gene Expression Omnibus (GEO)
database at NCBI
(www.ncbi.nlm.nih.gov/geo).
Importantly, the expression profile of the complemented M.
tuberculosis ΔsigF mutant strain
(8) was virtually
identical to that of the wild type at each of the three time points
studied (data not
shown).

Exponential-phase microarray
analysis.In exponential
growth phase (A600 = 0.6), 16 of the 38
underexpressed genes encode hypothetical unknown proteins (Table
2). Some genes with
relatively lower expression in the mutant strain are involved in fatty
acid and phospholipid metabolism, such as MT2304/Rv2244 (acpM,
which codes for an acyl carrier protein) and MT0928/Rv0905
(echA6, which codes for enoyl coenzyme A (enoyl-CoA)
hydratase/isomerase and in detoxification, such as MT2912/Rv2846c
(efpA, which codes for efflux protein) and MT4033/Rv3914
(trxC, which codes for thioredoxin). Of note the ahpC
gene (MT2503/Rv2428), which encoded alkyl hydroperoxidase, is strongly
down-regulated by loss of SigF in exponential phase. The M.
tuberculosis ahpC gene has been implicated in isoniazid resistance
(52,
63), as well as virulence
(22,
55,
62). SigF may also
regulate genes involved in protein folding such as MT3527/Rv3418c
(groES, which codes for 10-kDa chaperonin) and MT0265/Rv0251c
(hsp, which codes for an Hsp20
homologue).

Stationary-phase microarray
analysis.SigF has been shown
to be more highly expressed in stationary growth phase and during
stress conditions (17,
38). Therefore, it is not
surprising the microarray analysis revealed that more genes were
down-regulated in the ΔsigF mutant strain in
stationary phase and under growth stress conditions than in the
exponential growth phase (Tables
2 and
4). The underexpressed genes include those involved in energy metabolism
(such as electron transport, fermentation, anaerobic metabolism, and
polysaccharide synthesis and degradation), nucleotide synthesis, and
central intermediary metabolism (including oxidoreductases,
arylsulfatases, methyl transferases, acyltransferases, monooxygenase
flavin adenine dinucleotide [FAD] binding protein, and a
glutamine amidotransferase). This broad range of genes whose expression
is influenced in part by SigF supports its role as a stress-response
regulator.

Cell envelope genes.In stationary phase several genes
involved in the biosynthesis and structure of the cell envelope were
relatively down-regulated in the ΔsigF mutant strain,
as shown by microarray analysis, as were several involved in the
biosynthesis and degradation of surface polysaccharides and
lipopolysaccharides (Table
4). Examples include
pimB (MT0583/Rv0557), which is involved in lipoarabinomannan
biosynthesis (49), and
murB (MT0500/Rv0482), which is involved in cell wall formation
and peptidoglycan biosynthesis
(4).

Since the
microarray studies showed that in the absence of sigF a number
of polyketide synthase genes, including pks2
(13,
46), are underexpressed,
which might affect the structure of the cell envelope in M.
tuberculosis, we evaluated wild-type and mutant strains for the
presence of cell envelope sulfolipids. We found theΔ
sigF mutation conferred negative neutral red staining
(Fig.
3) to the M. tuberculosis strain, suggesting reduced synthesis of
cell-envelope associated sulfolipids
(39).

SigF
as a member of the M. tuberculosis regulatory gene
hierarchy.Microarray
analysis revealed that several regulatory genes are SigF dependent. The
expression of sigF itself is down-regulated in the absence of
functional SigF protein indicating that sigF expression is at
least in part autoregulated. Indeed, a promoter upstream of
sigF and the anti-sigma factor gene usfX preceding it
has been shown by in vitro transcription to be SigF dependent
(2). Moreover, the
expression of alternate sigma factor gene sigC also appears to
be SigF dependent (Table
4). sigC is one
of the most abundantly expressed sigma factor genes
(34), and a recent study
has shown that M. tuberculosis sigC controls a large
multifaceted regulon and is essential for lethality in the mouse model
(58). Additionally, SigF
appears to play a role in the expression of several other
repressor/activators from the MarR, GntR, and TetR family of DNA
binding regulators (Table
4). The finding of
overlapping transcriptional influence on other regulators suggests that
SigF participates in a hierarchical network of M. tuberculosis
gene regulation—an observation previously made for M.
tuberculosis SigH
(27,
35,
45), SigE
(1,
36), and SigC
(58).

Identification
of a SigF promoter recognition consensus sequence.As shown by the microarray analysis,
several genes were relatively underexpressed in theΔ
sigF mutant strain. We would expect some of these
genes to be directly regulated by SigF, while others may be indirectly
regulated. To search for shared promoter sequences, we used the Search
Pattern function in Tuberculist
(http://genolist.pasteur.fr/TubercuList/)
and the B. subtilis SigB and M. tuberculosis
SigF-dependent promoter sequence GTTTX17GGGTAT
for the gene upstream of sigF, i.e., usfX
or rsbW (MT3386, Rv3287c) recently determined by biochemical
approaches (2). We
identified several SigF-influenced genes which are preceded by
sequences similar to that in the usfX promoter. Analysis was
restricted to genes, downregulated in the microarray analysis, with a
clear-cut 5′-untranslated region rather than those that are
downstream members of an operon. Allowing no more than two mismatches
in each hexamer with a spacer size ranging from 16 to 20 nucleotides
(nt), putative promoter sequences were found in intergenic regions up
to 500 bp from the start site. Tables
5 and
6 show sets of genes repressed in the mutant in stationary phase with
either no more than three or a maximum of four, respectively, total
mismatches in the −35 and −10 regions compared to the
usfX promoter sequence. Restricting the number of mismatches
to three or fewer revealed a set of 14 genes whose expression was
downregulated in the ΔsigF mutant strain in the
microarray analysis and revealed a consensus sequence of −35
GGTTTC and −10 GGGTAT
(Table 5). The
consensus promoter for these 14 genes has a consensus spacer size of 18
nt with the GGG motif in the −10 region being absolutely
conserved. The distance from the translational start site to the
5′ end of the promoter region averages approximately 200 nt.
The stacking energy (measured on a scale of −3.82 kcal/mol for
TA dinucleotides to −14.59 kcal/mol for GC dinucleotides; see
btwisted at
http://bioinfo.pbi.nrc.ca:8090/EMBOSS/index.html)
in the −10 element of the promoter region is a relatively low−
7.55 kcal/mol/bp within the promoter unwinding region. The GC
content of the consensus promoter hexamers is 50%, notably lower
than the 66% found in the M. tuberculosis genome as a
whole.

Genes
having four mismatches in the −35 and −10 consensus
SigF promoter regions that were found to be down-regulated in
stationary phase in the M. tuberculosisΔ
sigF mutant strain

DISCUSSION

A previous study from
our group showed that the M. tuberculosisΔ
sigF mutant strain and its otherwise-isogenic
parental wild-type strain CDC1551 have the same growth rates both in
broth culture and during intracellular growth in human monocyte-derived
macrophages (8). However,
following intravenous infection in mice a time-to-death study showed
that the ΔsigF mutant strain was significantly
attenuated, with mouse survival times for mutant infection of up to 334
days (median time to death, 246 days) compared to 184 days (median time
to death, 161 days) for wild-type-infected mice
(8). In the present study,
we show that the ΔsigF mutant strain is capable of
early proliferation followed by persistence at high CFU counts in mouse
lungs for at least 20 weeks. While the ΔsigF mutant
strain persisted in lung tissues it did not achieve levels as high as
those observed with wild-type infection, which exceeded 7.7 ×
106 CFU when BALB/c mice were used. The histopathology of
the lung and spleen confirms that there is less disease progression in
the mice infected with the mutant strain. We did not assess the
phenotype of the complemented ΔsigF mutant strain in
this organ CFU count assay; however, previous studies have indicated
that sigF complementation restores wild-type levels of
resistance to rifampin and wild-type levels of chenodeoxycholate uptake
(8). Moreover, in the
present study we observed that sigF complementation reverses
the loss of neutral red staining seen in the ΔsigF
mutant strain and also restores the microarray gene expression pattern
of the ΔsigF mutant strain to one virtually identical
to that of wild type. Thus, complementation of sigF restores
multiple phenotypes observed in the ΔsigF mutant
strain back to their wild-type state, indicating that the mutant
phenotypes observed are due solely to replacement of the sigF
gene and not some unanticipated second site mutation. In contrast to
the ΔsigF mutant strain, the pattern of infection by
M. bovis BCG, an attenuated strain widely used as a
tuberculosis vaccine, showed a pattern similar to that of so-called
persistence (per) mutants
(37) with initial
proliferation in lungs for 2 to 3 weeks followed by gradual host
clearance in BALB/c mice (data not shown).

This study taken
together with the earlier one by Chen et al.
(8) reveals that the
M. tuberculosis ΔsigF mutant strain achieves
and persists at high colony counts in mouse tissues but is attenuated
in eliciting histopathologic evidence of tissue damage and in producing
lethality in mice. This general pattern of attenuation (high lung CFU
counts but delayed time to death) has been referred to as the
immunopathology phenotype (Imp or Pat
[23]) and has
been observed in three other M. tuberculosis alternate sigma
factor mutants—the M. tuberculosisΔ
sigH
(27),ΔsigE
(1), andΔ
sigC
(58) mutants.
Additionally, the immunopathology pattern of attenuation was noted in
M. tuberculosis lacking the whiB3 gene
(57) and the RD1 region
of deletion (29). Since
there is evidence that RD1 contains regulatory genes
(31) and that WhiB3 is a
regulator of RNA polymerase
(57), the immunopathology
phenotype appears to result from several diverse defects in the M.
tuberculosis regulatory apparatus. It is noteworthy that the
expression of sigC was reduced in the M. tuberculosisΔ
sigF mutant strain. Since both theΔ
sigF and the ΔsigC mutant strains
have been observed to display the immunopathology phenotype in mice, it
is conceivable that these two sigma factors form a regulatory hierarchy
which governs the expression of a key group of immunopathology
antigens. To date no specific effector genes or proteins of the
immunopathology phenotype have been identified which might account for
failure of this class of M. tuberculosis mutants to elicit
wild-type levels of tissue damage in the absence of a clear-cut
bacterial survival defect.

Because of its ability to persist but
not elicit the same degree of lethality, we tested the M.
tuberculosis ΔsigF mutant strain as a
tuberculosis vaccine using the rabbit aerosol challenge model with
tubercle counting as the efficacy parameter. The rabbit is an important
model for vaccine testing against tuberculosis because of the
similarities between the rabbit and human forms of disease
(15). After aerosol
challenge with M. tuberculosis, the ability of various
vaccines to prevent grossly visible primary tubercles has allowed us to
differentiate the relative efficacy of candidate vaccines
(6,
14). Using this model we
observed fewer tubercles in the lungs of rabbits vaccinated
intradermally with the ΔsigF mutant strain than in
either rabbits vaccinated with BCG or unvaccinated controls. The
protection conferred by the ΔsigF mutant strain
approached statistical significance (P = 0.06) in this
experiment using relatively small groups of outbred rabbits. Hence, in
the rabbit model, the M. tuberculosis ΔsigF
mutant strain confers some protection against tubercle formation after
infection with M. tuberculosis. The M. tuberculosisΔ
sigF mutant strain is among the first
immunopathology-type of mutant to be evaluated as a live attenuated
vaccine against tuberculosis. Hsu et al.
(24) reported that aΔ
RD1 M. tuberculosis mutant conferred protection
similar to that observed for BCG, all strains of which lack the RD1
region. Interestingly, they also found that the loss of cytolytic
activity for pneumocytes coincided with the deletion of a more specific
region, namely, the deletion of the Rv3874/Rv3875 (cfp
10/esat-6 or esxB/esxA) segment of RD1.
These encouraging results suggest that the immunopathology mutant
class, with its unique ability to persist without aggressive
pathological tissue damage, may be useful in future vaccination
strategies for tuberculosis. This may be attributable to the presence
of important immunogenic proteins still present in these attenuated
immunopathology phenotype (Imp) mutants and/or their greater
persistence in tissues (7,
27). Whether these
mutants also persist after sensitization with environmental
mycobacteria remains to be tested. Studies to test the M.
tuberculosis ΔsigF mutant strain and other
immunopathology mutants both as stand-alone vaccines or as boosters to
previously administered BCG vaccines are under way in other animal
models.

Given the importance of SigF in mycobacterial responses
to stress and its increased expression in stationary phase, the finding
that the overwhelming majority of underexpressed genes are detected in
stationary phase is not surprising and further supports earlier studies
of this sigma factor (8,
16,
17,
38). In this study we
also identified a potentially significant in vitro phenotype of the
M. tuberculosis ΔsigF mutant strain, namely,
its inability to retain the neutral red stain which earlier studies
have found to correlate with a reduction in the expression of
envelope-associated mycobacterial sulfolipids (Fig.
3)
(39). Chen et al.
(8) noted that theΔ
sigF mutant strain was hypersusceptible to rifampin
and rifapentine and that its envelope showed permeability differences
to radiolabeled chenodeoxycholate. That cell envelope gene expression
is affected by the ΔsigF mutation may be further
evidence that SigF influences the expression of genes involved in the
structure and function of the mycobacterial cell wall and its complex
network of lipids and polysaccharides, including virulence-related
sulfolipids (20). The
cell envelope of M. tuberculosis has long been identified as a
defense against chemical injury, dehydration, and certain antibiotics
(26,
30,
59). This is due in part
to the low permeability of the unique sacculus and to small hydrophilic
molecules. The relative underexpression of certain membrane proteins,
lipoproteins, surface polysaccharides, lipopolysaccharides, and murein
sacculus could affect the permeability of the cell wall and alter the
antigenic profile of the ΔsigF mutant during
infection. The ΔsigF mutant strain also shows reduced
expression of several key regulators some of which (TetR, GntR, and
MarR) govern the expression of efflux pumps in other bacteria
(9,
50,
51) which could play a
role in the novel immunopathology phenotype observed in the
host-pathogen interaction of the ΔsigF mutant strain
in mice. Loss of SigF was also found to reduce the expression of the
sigma factor SigC. Taken together these observations suggest that SigF
governs several subordinate regulons.

Sigma factors are
associated with RNA polymerase and direct the recognition of DNA
promoter sequences. Identification of promoter sequences based on
functional or structural homology to sigma factors in other bacteria
assumes a conservation not only of the sigma factor structural genes
but in DNA recognition sites as well. Such selection pressure in
noncoding promoter-containing sequences may not, however, be as intense
as that in coding regions for the sigma factor genes themselves. The
availability of a biochemically identified promoter sequence in the
adjacent anti-SigF gene, usfX or rsbW, which is
itself regulated by SigF
(2), combined with our
microarray results, allowed us to use pattern search analysis to seek
additional genes likely to be regulated directly by this sigma factor.
Indeed, we found a number of genes regulated by SigF in stationary
phase that contain potential promoter sequences related to that found
for the control of the usfX-sigF operon, and this permitted us
to derive a putative SigF consensus recognition sequence,
GGTTTCX18GGGTAT. This putative M.
tuberculosis SigF promoter consensus sequence is remarkably
similar to that for the B. subtilis stationary-phase and
stress response sigma factor, SigB, which has GTTT in the −35
hexamer and GGGWAW (W = T or A) in the−
10 region (43,
44). The conservation of
the putative M. tuberculosis SigF −35 hexamer ranges
from 43 to 93%, suggesting that certain residues may be more
critical for recognition than others. In the regulon set reported here,
the GGG motif is 100% conserved in the −10 region
hexamer, while the remaining residues are 57, 64, and 71%
conserved. For the −10 and −35 hexamers, no putative
promoter sequence was less than 67% conserved, possibly
indicating that the presence of at least four of the residues in each
hexamer—at an appropriate distance from the other
hexamer—is sufficient to permit SigF recognition. In addition,
the variability of the promoter sequences within the same organism
suggests that extrapolation of conservation from organisms with
different GC content may not be a strong approach to promoter
identification. However, as a prelude to direct biochemical assessment,
homology-based searches remain a prudent and efficient search method
for identifying putative members of a regulon.

In summary, we
have shown that the ΔsigF mutant strain belongs to the
newly appreciated class of mutants displaying defective immunopathology
during mouse infection and reduced lethality in time-to-death assays,
but with high-level bacterial persistence in mouse lung. The high
degree of survival in tissues with reduced lethality suggests that this
class of mutant may be valuable as live-attenuated vaccines for
tuberculosis. Indeed, the ΔsigF mutant strain appears
to confer somewhat stronger immunity than BCG against virulent M.
tuberculosis challenge in the rabbit model. If this finding is
confirmed in the mouse and possibly guinea pig models, it may suggest
that alterations in the SigF regulon result in the presentation of a
superior set of immunogens than BCG with potentially more long-lived
protection. The microarray analysis reported here represents an initial
characterization of that regulon.

ACKNOWLEDGMENTS

This work was supported by
grants from the NIH (AI36973, AI37856, and AI43846), the National
Vaccine Program Office, and the Sequella Global Tuberculosis
Foundation.

The technical assistance of Rafael Ruiz and editorial
help by Naomi Gauchet are gratefully acknowledged. We are grateful to
M. Louise M. Pitt of USAMRIID and Bernard Zook of George Washington
University for their generous assistance with rabbit
experiments.

Stead,
W. W.1967. Pathogenesis of a first episode
of chronic pulmonary tuberculosis in man: recrudescence of residuals of
the primary infection or exogenous reinfection? Am. Rev. Respir.
Dis.95:729-745.